PsbQ (Sll1638) in Synechocystis sp. PCC 6803 is required for photosystem II activity in specific mutants and in nutrient-limiting conditions

A PsbQ homologue has been found associated with photosystem II complexes in Synechocystis sp. PCC 6803 where it is involved in optimal photoautotrophic growth and water splitting under CaCl(2)-depleted conditions [Thornton, L. E., Ohkawa, H., Roose, J. L., Kashino, Y., Keren, N., and Pakrasi, H. B. (2004) Plant Cell 16, 2164-2175]. By inactivating psbQ in strains carrying photosystem II-specific mutations, we have identified stringent requirements for PsbQ in vivo. Whereas under nutrient-replete conditions the DeltaPsbQ mutant was similar to wild type, a strain lacking PsbQ and PsbV was not photoautotrophic, exhibiting decreased oxygen evolution and decreased photosystem II assembly compared to the DeltaPsbV mutant. Combining the removal of PsbU and PsbQ introduced an altered requirement for Ca(2+) and Cl(-), and photoautotrophic growth of the DeltaPsbQ strain was prevented in nutrient-limiting media depleted in Ca(2+), Cl(-), and iron. Unlike other photosystem II extrinsic proteins PsbQ did not participate in the acquisition of thermotolerance; however, photoautotrophic growth at elevated temperatures was impaired in this mutant. Growth of the DeltaPsbV:DeltaPsbQ mutant was restored at pH 10.0: in contrast, an additional deletion between Arg-384 and Val-392 in the CP47 protein of photosystem II prevented recovery at alkaline pH. When conditions prevented photoautotrophy in strains lacking PsbQ, photoheterotrophic growth was indistinguishable to wild type, indicating that photosystem II had been inactivated. These data substantiate a role for PsbQ in optimizing photosystem II activity in Synechocystis sp. PCC 6803 and establish an absolute requirement for the subunit under specific biochemical and physiological conditions.     

Requirements for different combinations of the extrinsic proteins in specific cyanobacterial Photosystem II mutants

The crystallographic data available for Photosystem II (PS II) in cyanobacteria has now provided complete structures for loop E from CP43 and CP47 as well as the extrinsic subunits PsbO, PsbU and PsbV. Protein interactions between these subunits are essential for stable water splitting and there is evidence that the binding of PsbU facilitates optimal energy transfer from the phycobilisome. Interactions between PsbO and CP47 may also play a role in dimer stabilization while loop E of CP43 contributes directly to the water-splitting reaction. Recent evidence also suggests that homologs of PsbP and PsbQ play key roles in cyanobacterial PS II, and under nutrient-deficient conditions PsbQ appears essential for photoautotrophic growth.     

Homologs of plant PsbP and PsbQ proteins are necessary for regulation of photosystem ii activity in the cyanobacterium Synechocystis 6803

The mechanism of oxygen evolution by photosystem II (PSII) has remained highly conserved during the course of evolution from ancestral cyanobacteria to green plants. A cluster of manganese, calcium, and chloride ions, whose binding environment is optimized by PSII extrinsic proteins, catalyzes this water-splitting reaction. The accepted view is that in plants and green algae, the three extrinsic proteins are PsbO, PsbP, and PsbQ, whereas in cyanobacteria, they are PsbO, PsbV, and PsbU. Our previous proteomic analysis established the presence of a PsbQ homolog in the cyanobacterium Synechocystis 6803. The current study additionally demonstrates the presence of a PsbP homolog in cyanobacterial PSII. Both psbP and psbQ inactivation mutants exhibited reduced photoautotrophic growth as well as decreased water oxidation activity under CaCl(2)-depleted conditions. Moreover, purified PSII complexes from each mutant had significantly reduced activity. In cyanobacteria, one PsbQ is present per PSII complex, whereas PsbP is significantly substoichiometric. These findings indicate that both PsbP and PsbQ proteins are regulators that are necessary for the biogenesis of optimally active PSII in Synechocystis 6803. The new picture emerging from these data is that five extrinsic PSII proteins, PsbO, PsbP, PsbQ, PsbU, and PsbV, are present in cyanobacteria, two of which, PsbU and PsbV, have been lost during the evolution of green plants.     

The Psb27 protein facilitates manganese cluster assembly in photosystem II

Photosystem II (PSII) is a large membrane protein complex that uses light energy to convert water to molecular oxygen. This enzyme undergoes an intricate assembly process to ensure accurate and efficient positioning of its many components. It has been proposed that the Psb27 protein, a lumenal extrinsic subunit, serves as a PSII assembly factor. Using a psb27 genetic deletion strain (Deltapsb27) of the cyanobacterium Synechocystis sp. PCC 6803, we have defined the role of the Psb27 protein in PSII biogenesis. While the Psb27 protein was not essential for photosynthetic activity, various PSII assembly assays revealed that the Deltapsb27 mutant was defective in integration of the Mn(4)Ca(1)Cl(x) cluster, the catalytic core of the oxygen-evolving machinery within the PSII complex. The other lumenal extrinsic proteins (PsbO, PsbU, PsbV, and PsbQ) are key components of the fully assembled PSII complex and are important for the water oxidation reaction, but we propose that the Psb27 protein has a distinct function separate from these subunits. We show that the Psb27 protein facilitates Mn(4)Ca(1)Cl(x) cluster assembly in PSII at least in part by preventing the premature association of the other extrinsic proteins. Thus, we propose an exchange of lumenal subunits and cofactors during PSII assembly, in that the Psb27 protein is replaced by the other extrinsic proteins upon assembly of the Mn(4)Ca(1)Cl(x) cluster. Furthermore, we show that the Psb27 protein provides a selective advantage for cyanobacterial cells under conditions such as nutrient deprivation where Mn(4)Ca(1)Cl(x) cluster assembly efficiency is critical for survival.     

PsbU provides a stable architecture for the oxygen-evolving system in cyanobacterial photosystem II

PsbU is a lumenal peripheral protein in the photosystem II (PS II) complex of cyanobacteria and red algae. It is thought that PsbU is replaced functionally by PsbP or PsbQ in plant chloroplasts. After the discovery of PsbP and PsbQ homologues in cyanobacterial PS II [Thornton et al. (2004) Plant Cell 16, 2164-2175], we investigated the function of PsbU using a psbU deletion mutant (DeltaPsbU) of Synechocystis 6803. In contrast to the wild type, DeltaPsbU did not grow when both Ca2+ and Cl- were eliminated from the growth medium. When only Ca2+ was eliminated, DeltaPsbU grew well, whereas when Cl- was eliminated, the growth rate was highly suppressed. Although DeltaPsbU grew normally in the presence of both ions under moderate light, PS II-related disorders were observed as follows. (1) The mutant cells were highly susceptible to photoinhibition. (2) Both the efficiency of light utilization under low irradiance and the chlorophyll-specific maximum rate of oxygen evolution in DeltaPsbU cells were 60% lower than those of the wild type. (3) The decay of the S2 state in DeltaPsbU cells was decelerated. (4) In isolated PS II complexes from DeltaPsbU cells, the amounts of the other three lumenal extrinsic proteins and the electron donation rate were drastically decreased, indicating that the water oxidation system became significantly labile without PsbU. Furthermore, oxygen-evolving activity in DeltaPsbU thylakoid membranes was highly suppressed in the absence of Cl-, and 60% of the activity was restored by NO3- but not by SO4(2-), indicating that PsbU had functions other than stabilizing Cl-. On the basis of these results, we conclude that PsbU is crucial for the stable architecture of the water-splitting system to optimize the efficiency of the oxygen evolution process.     

Refinement of the structural model for the Photosystem II supercomplex of higher plants

Recent X-ray structures determined for the Photosystem II (PSII) core complex isolated from cyanobacteria have provided important information for understanding the functionality of this photosynthetic enzyme including its water splitting activity. As yet, no high-resolution structure is available for PSII of plants or eukaryotes in general. However, crystal structures have been determined for some components of plant PSII which together with the cyanobacterial structure can be used to interpret lower resolution structures of plant PSII derived from electron cryomicroscopy (cryo-EM). Here, we utilise the published X-ray structures of a cyanobacterial PSII core, Light Harvesting Complex II (LHCII), PsbP and PsbQ proteins to construct a model of the plant LHCII-PSII supercomplex using a 17 A resolution 3D electron density map of the spinach supercomplex determined by cryo-EM and single particle analysis. In so doing, we tentatively identify the relative positioning of the chlorophylls within the supercomplex and consider energy transfer pathways between the different subunits. The modelling has also allowed density to be assigned to the three extrinsic proteins of plant PSII, PsbO, PsbP and PsbQ associated with the water splitting centre and concluded that although the position of PsbO is the same as in cyanobacteria, PsbP and PsbQ are located in different positions to the cyanobacterial extrinsic PsbU and PsbV proteins.     

The PsbP protein, but not the PsbQ protein, is required for normal thylakoid architecture in Arabidopsis thaliana

Interfering RNA was used to suppress the expression of the genes At1g06680 and At2g30790 in Arabidopsis thaliana, which encode the PsbP-1 and PsbP-2 proteins, respectively, of Photosystem II. A phenotypic series of transgenic plants was recovered that expressed intermediate and low amounts of PsbP. Earlier we had documented significant alterations in a variety of Photosystem II parameters in these plant lines [Yi, X., Liu, H., Hargett, S. R., Frankel, L. K., Bricker, T. M. (2007). The PsbP protein is required for photosystem II complex assembly/stability and photoautotrophy in Arabidopsis thaliana. J. Biol. Chem. 34, 24833-24841]. In this communication, we document extensive defects in the thylakoid membrane architecture of these plants. Interestingly, strong interfering RNA suppression of the genes encoding the PsbQ protein (At4g21280 and At4g05180) was found to have no effect on the architecture of thylakoid membranes.     

The PsbQ protein stabilizes the functional binding of the PsbP protein to photosystem II in higher plants

PsbP and PsbQ proteins are extrinsic subunits of photosystem II (PSII) and optimize the oxygen evolution reaction by regulating the binding properties of the essential cofactors Ca(2+) and Cl(-). PsbP induces conformational changes around the catalytic Mn cluster required for Ca(2+) and Cl(-) retention, and the N-terminal region of PsbP is essential for this reaction. It was reported that PsbQ partially restores the functional defect of N-terminal truncated PsbP [Ifuku and Sato (2002) Plant Cell Physiol. 43, 1244-1249]; however, the mechanism of this restoration is yet to be clarified. In this study, we demonstrate that PsbQ is able to restore the functional binding of mutated PsbPs. In the presence of PsbQ, ?15-PsbP, a truncated PsbP lacking 15 N-terminal residues, was able to specifically bind to NaCl-washed spinach PSII membranes and significantly restore the oxygen evolving activity. Furthermore, PsbQ was also able to compensate for the impaired ion-retention of H144A-PsbP, in which a conserved histidine at position 144 in the C-terminal domain was substituted with an alanine. Fourier transform infrared (FTIR) difference spectroscopy showed that PsbQ restored the ability of ?15- and H144A-PsbP to induce proper conformational changes during S(1) to S(2) transition. These data suggest that the major function of PsbQ is to stabilize PsbP binding, thereby contributing to the maintenance of the catalytic Mn cluster of the water oxidation machinery in higher plant PSII. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Investigation of a requirement for the PsbP-like protein in Synechocystis sp. PCC 6803

The sll1418 gene encodes a PsbP-like protein in Synechocystis sp. PCC 6803. Expression of sll1418 was similar in BG-11 and in Cl⁻- or Ca²⁺-limiting media, and inactivation of sll1418 did not prevent photoautotrophic growth in normal or nutrient-limiting conditions. Also the wild-type and ΔPsbP strains exhibited similar oxygen evolution and assembly of Photosystem II (PS II) centers. Inactivation of sll1418 in the ΔPsbO: ΔPsbP, ΔPsbQ:ΔPsbP, ΔPsbU:ΔPsbP and ΔPsbV:ΔPsbP mutants did not prevent photoautotrophy or alter PS II assembly and oxygen evolution in these strains. Moreover, the absence of PsbP did not affect the ability of alkaline pH to restore photoautotrophic growth in the ΔPsbO:ΔPsbU strain. The PsbO, PsbU and PsbV proteins are required for thermostability of PS II and thermal acclimation in Synechocystis sp. PCC 6803 [Kimura et al. (2002) Plant Cell Physiol 43: 932–938]. However, thermostability and thermal acclimation in ΔPsbP cells were similar to wild type. These results are consistent with the conclusion that PsbP is associated with ∼3 of PS II centers, and may play a regulatory role in PS II [Thornton et al. (2004) Plant Cell 16: 2164–2175].     

Structurally conserved channels in cyanobacterial and plant photosystem II

In the cyanobacterial photosystem II (PSII), the O4-water chain in the D1 and CP43 proteins, a chain of water molecules that are directly H-bonded to O4 of the Mn₄Ca cluster, is linked with a channel that connects the protein bulk surface along with a membrane-extrinsic protein subunit, PsbU (O4-PsbU channel). The cyanobacterial PSII structure also shows that the O1 site of the Mn₄Ca cluster has a chain of H-bonded water molecules, which is linked with the channel that proceeds toward the bulk surface via PsbU and PsbV (O1-PsbU/V channel). Membrane-extrinsic protein subunits PsbU and PsbV in cyanobacterial PSII are replaced with PsbP and PsbQ in plant PSII. However, these four proteins have no structural similarity. It remains unknown whether the corresponding channels also exist in plant PSII, because water molecules are not identified in the plant PSII cryo-electron microscopy (cryo-EM) structure. Using the cyanobacterial and plant PSII structures, we analyzed the channels that proceed from the Mn₄Ca cluster. The cyanobacterial O4-PsbU and O1-PsbU/V channels were structurally conserved as the channel that proceeds along PsbP toward the protein bulk surface in the plant PSII (O4-PsbP and O1-PsbP channels, respectively). Calculated protonation states indicated that in contrast to the original geometry of the plant cryo-EM structure, protonated PsbP-Lys166 may form a salt-bridge with ionized D1-Glu329 and protonated PsbP-Lys173 may form a salt-bridge with ionized PsbQ-Asp28 near the O1-PsbP channel. The existence of these channels might explain the molecular mechanism of how PsbP can interact with the Mn₄Ca cluster.     

The PsbQ′ protein affects the redox potential of the Q<Subscript>A</Subscript> in photosystem II

Red alga contains four extrinsic proteins in photosystem II (PSII), which are PsbO, PsbV, PsbU, and PsbQ′. Except for the PsbQ′, the composition is the same in cyanobacterial PSII. Reconstitution analysis of cyanobacterial PSII has shown that oxygen-evolving activity does not depend on the presence of PsbQ′. Recently, the structure of red algal PSII was elucidated. However, the role of PsbQ′ remains unknown. In this study, the function of the acceptor side of PSII was analyzed in PsbQ′-reconstituted PSII by redox titration of Q_A and thermoluminescence. The redox potential of Q_A was positively shifted when PsbQ′ was attached to the PSII. The positive shift of Q_A is thought to cause a decrease in the amount of triplet chlorophyll in PSII. On the basis of these results, we propose that PsbQ′ has a photoprotective function when irradiated with strong light.     

Extrinsic proteins of photosystem II: an intermediate member of PsbQ protein family in red algal PS II.

The oxygen-evolving photosystem II (PS II) complex of red algae contains four extrinsic proteins of 12 kDa, 20 kDa, 33 kDa and cyt c-550, among which the 20 kDa protein is unique in that it is not found in other organisms. We cloned the gene for the 20-kDa protein from a red alga Cyanidium caldarium. The gene consists of a leader sequence which can be divided into two parts: one for transfer across the plastid envelope and the other for transfer into thylakoid lumen, indicating that the gene is encoded by the nuclear genome. The sequence of the mature 20-kDa protein has low but significant homology with the extrinsic 17-kDa (PsbQ) protein of PS II from green algae Volvox Carteri and Chlamydomonas reinhardtii, as well as the PsbQ protein of higher plants and PsbQ-like protein from cyanobacteria. Cross-reconstitution experiments with combinations of the extrinsic proteins and PS IIs from the red alga Cy. caldarium and green alga Ch. reinhardtii showed that the extrinsic 20-kDa protein was functional in place of the green algal 17-kDa protein on binding to the green algal PS II and restoration of oxygen evolution. From these results, we conclude that the 20-kDa protein is the ancestral form of the extrinsic 17-kDa protein in green algal and higher plant PS IIs. This provides an important clue to the evolution of the oxygen-evolving complex from prokaryotic cyanobacteria to eukaryotic higher plants. The gene coding for the extrinsic 20-kDa protein was named psbQ' (prime).     

Crystal structure of the PsbP protein of photosystem II from Nicotiana tabacum

PsbP is a membrane-extrinsic subunit of the water-oxidizing complex photosystem II (PS II). The evolutionary origin of PsbP has long been a mystery because it specifically exists in higher plants and green algae but not in cyanobacteria. We report here the crystal structure of PsbP from Nicotiana tabacum at a resolution of 1.6 Å. Its structure is mainly composed of β-sheet, and is not similar to any structures in cyanobacterial PS II. However, the electrostatic surface potential of PsbP is similar to that of cyanobacterial PsbV (cyt c₅₅₀), which has a function similar to PsbP. A structural homology search with the DALI algorithm indicated that the folding of PsbP is very similar to that of Mog1p, a regulatory protein for the nuclear transport of Ran GTPase. The structure of PsbP provides insight into its novel function in GTP-regulated metabolism in PS II.     

The PsbQ protein is required in Arabidopsis for photosystem II assembly/stability and photoautotrophy under low light conditions

RNA interference was used to simultaneously suppress the expression of the two genes that encode the PsbQ proteins of Photosystem II (PS II) in Arabidopsis thaliana, psbQ-1 (At4g21280) and psbQ-2 (At4g05180). Two independent PsbQ-deficient plant lines were examined. These plant lines produced little detectable PsbQ protein. Under normal growth light conditions, the wild type and mutant plants were visually indistinguishable. Additionally, analysis of steady state oxygen evolution rates and chlorophyll fluorescence characteristics indicated little alteration of photosynthetic capacity in the mutant plants. No loss of other PS II proteins was evident. Interestingly, flash oxygen yield analysis performed on thylakoid membranes isolated from the mutant and wild type plants indicated that the oxygen-evolving complex was quite unstable in the mutants. Furthermore, the lifetime of the S2 state of the oxygen-evolving complex appeared to be increased in these plants. Incubation of the wild type and mutant plants under low light growth conditions led to a significantly stronger observed phenotype in the mutants. The mutant plants progressively yellowed (after 2 weeks) and eventually died (after 3-4 weeks). The wild type plants exhibited only slight yellowing after 4 weeks under low light conditions. The mutant plants exhibited a large loss of a number of PS II components, including CP47 and the D2 protein, under low light conditions. Additionally, significant alterations of their fluorescence characteristics were observed, including an increased FO and decreased FV, yielding a large loss in PS II quantum efficiency (FV/FM). Analysis of QA- decay kinetics in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea indicated a defect in electron transfer from QA- to QB, whereas experiments performed in the presence of this herbicide indicated that the recombination rate between QA- and the S2 state was strongly retarded. These results indicate that the loss of the PsbQ protein induces significant changes in Photosystem II function, particularly in low light-grown plants, and that the PsbQ protein is required for photoautotrophic growth under low light conditions.     

Subcellular localization of the BtpA protein in the cyanobacterium Synechocystis sp. PCC 6803.

Photosystem I is a large pigment-protein complex embedded in the thylakoid membranes of chloroplasts and cyanobacteria. In the cyanobacterium Synechocystis sp. PCC 6803, the btpA gene encodes a 30-kDa polypeptide. Mutations in this gene significantly affect accumulation of the reaction center proteins of photosystem I in Synechocystis 6803 [Bartsevich, V. V. & Pakrasi, H. B. (1997) J. Biol. Chem. 272, 6372-6378]. We describe here the intracellular localization of the BtpA protein. Immunolocalization in Synechocystis 6803 cells demonstrated that the BtpA protein is tightly associated with the thylakoid membranes. Phase fractionation in the detergent Triton X-114 indicated that BtpA is a peripheral membrane protein. To determine which surface of the thylakoid membrane BtpA is exposed to, we used a two-phase polymer partitioning technique to develop a novel method to isolate inside-out and right-side-out thylakoid vesicles from Synechocystis 6803. Treatments of such vesicles with different salts and protease showed that the BtpA protein is an extrinsic membrane protein which is exposed to the cytoplasmic face of the thylakoid membrane.     

Functional characterization of monomeric photosystem II core preparations from Thermosynechococcus elongatus with or without the Psb27 protein

Two monomeric fractions of photosystem II (PS II) core pacticles from the thermophilic cyanobacterium Thermosynechococcus elongatus have been investigated using flash-induced variable fluorescence kinetics and EPR spectroscopy. One fraction was highly active in oxygen evolution and contained the extrinsic protein subunits PsbO, PsbU, and PsbV. The other monomeric fraction lacked oxygen evolving activity as well as the three extrinsic subunits, but the luminally located, extrinsic Psb27 lipoprotein was present. In the monomeric fraction with bound Psb27, flash-induced variable fluorescence showed an absence of oxidizable Mn on the donor side of PS II and impaired forward electron transfer from the primary quinone acceptor, QA. These results were confirmed with EPR spectroscopy by the absence of the "split S1" interaction signal from YZ* and the CaMn4 cluster and by the absence of the S2-state multiline signal. A different protein composition on the donor side of PS II monomers with Psb27 was also supported by the lack of an EPR signal from cytochrome c550 (in the PsbV subunit). In addition, we did not observe any oxidation of cytochrome b559 at low temperature in this fraction. The presence of Psb27 and the absence of the CaMn4 cluster did not affect the protein matrix around YD or the acceptor side quinones as can be judged from the appearance of the corresponding EPR signals. The diminished electron transport capabilities on both the donor and the acceptor side of PS II when Psb27 is present give further indications that this PS II complex is involved in the earlier steps of the PS II repair cycle.     

Three-dimensional structure of Chlamydomonas reinhardtii and Synechococcus elongatus photosystem II complexes allows for comparison of their oxygen-evolving complex organization

Electron microscopy and single-particle analyses have been carried out on negatively stained photosystem II (PSII) complexes isolated from the green alga Chlamydomonas reinhardtii and the thermophilic cyanobacterium Synechococcus elongatus. The analyses have yielded three-dimensional structures at 30-A resolution. Biochemical analysis of the C. reinhardtii particle suggested it to be very similar to the light-harvesting complex II (LHCII).PSII supercomplex of spinach, a conclusion borne out by its three-dimensional structure. Not only was the C. reinhardtii LHCII.PSII supercomplex dimeric and of comparable size and shape to that of spinach, but the structural features for the extrinsic OEC subunits bound to the lumenal surface were also similar thus allowing identification of the PsbO, PsbP, and PsbQ OEC proteins. The particle isolated from S. elongatus was also dimeric and retained its OEC proteins, PsbO, PsbU, and PsbV (cytochrome c(550)), which were again visualized as protrusions on the lumenal surface of the complex. The overall size and shape of the cyanobacterial particle was similar to that of a PSII dimeric core complex isolated from spinach for which higher resolution structural data are known from electron crystallography. By building the higher resolution structural model into the projection maps it has been possible to relate the positioning of the OEC proteins of C. reinhardtii and S. elongatus with the underlying transmembrane helices of other major intrinsic subunits of the core complex, D1, D2, CP47, and CP43 proteins. It is concluded that the PsbO protein is located over the CP47 and D2 side of the reaction center core complex, whereas the PsbP/PsbQ and PsbV/PsbU are positioned over the lumenal surface of the N-terminal region of the D1 protein. However, the mass attributed to PsbV/PsbU seems to bridge across to the PsbO, whereas the PsbP/PsbQ proteins protrude out more from the lumenal surface. Nevertheless, within the resolution and quality of the data, the relative positions of the center of masses for OEC proteins of C. reinhardtii and S. elongatus are similar and consistent with those determined previously for the OEC proteins of spinach.     

Kojic Acid Oxidase

Photosystem II (PSII), which catalyzes photosynthetic water oxidation, is composed of more than 20 subunits, including membrane-intrinsic and -extrinsic proteins. The extrinsic proteins of PSII shield the catalytic Mn₄CaO₅ cluster from exogenous reductants and serve to optimize oxygen evolution at physiological ionic conditions. These proteins include PsbO, found in all oxygenic organisms, PsbP and PsbQ, specific to higher plants and green algae, and PsbU, PsbV, CyanoQ, and CyanoP in cyanobacteria. Furthermore, red algal PSII has PsbQ′ in addition to PsbO, PsbV, and PsbU, and diatoms have Psb31 in supplement to red algal-type extrinsic proteins, exemplifying the functional divergence of these proteins during evolution. This review provides an updated summary of recent findings on PSII extrinsic proteins and discusses their binding, function, and evolution within various photosynthetic organisms.     

Deletion Mutagenesis of the Cytochrome b559 Protein Inactivates the Reaction Center of Photosystem II

In green plant-like photosynthesis, oxygen evolution is catalyzed by a thylakoid membrane-bound protein complex, photosystem II. Cytochrome b559, a protein component of the reaction center of this complex, is absent in a genetically engineered mutant of the cyanobacterium, Synechocystis 6803 [Pakrasi, H.B., Williams, J.G.K., and Arntzen, C.J. (1988). EMBO J. 7, 325-332]. In this mutant, the genes psbE and psbF, encoding cytochrome b559, were deleted by targeted mutagenesis. Two other protein components, D1 and D2 of the photosystem II reaction center, are also absent in this mutant. However, two chlorophyll-binding proteins, CP47 and CP43, as well as a manganese-stabilizing extrinsic protein component of photosystem II are stably assembled in the thylakoids of this mutant. Thus, this deletion mutation destabilizes the reaction center of photosystem II only. The mutant also lacks a fluorescence maximum peak at 695 nm (at 77 K) even though the CP47 protein, considered to be the origin of this fluorescence peak, is present in this mutant. We propose that the fluorescence at 695 nm originates from an interaction between the reaction center of photosystem II and CP47. The deletion mutant shows the absence of variable fluorescence at room temperature, indicating that its photosystem II complex is photochemically inactive. Also, photoreduction of QA, the primary acceptor quinone in photosystem II, could not be detected in the mutant. We conclude that cytochrome b559 plays at least an essential structural role in the reaction center of photosystem II.     

Uncoupling of detectable O2 evolution from the apparent S-state transitions in Photosystem II by lauroylcholine chloride: Possible implications in the photosynthetic water-splitting mechanism

Recently we have introduced the use of choline / fatty acid derived compounds, in particular lauroylcholine chloride (LCC), to probe selectively Photosystem II (PS II) structure and function (Wydrzynski, T. and Huggins, B.J. (1983) in The Oxygen-Evolving System of Photosynthesis (Inoue, Y., Crofts, A.R., Govindjee, Murata, N., Renger, G. and Satoh, K., eds.), pp. 265–272, Academic Press Tokyo, Japan). In this paper we report an unusual condition in thylakoid membrane samples at relatively low amounts of LCC in which detectable O₂ evolution cannot be measured, yet electron flow through PS II is near normal without added electron donors. LCC does not appear to interfere with the O₂ yield measurements directly nor act as an electron donor itself after the Tris block. Under this condition, steady state and flash O₂ yield measurements show no O₂ release or uptake, while steady-state ferricyanide photoreduction and the variable component of the chlorophyll a fluorescence transient remains at more than 50% of the control. The photoreduction of the primary quinone acceptor, Q_A, measured by microsecond range chlorophyll a fluorescence continues for a minimum of 200 single turnover excitation light flashes. Most importantly, the yield of the 35 |gms component of the chlorophyll a delayed fluorescence remains at approx. 65% of the control and oscillates with a normal period four over two cycles, indicating the normal cycling of the S-state transitions in PS II. Thus, it appears that PS II can operate normally without detectable O₂ evolution. The question remains as to whether water is still being photooxidized under this condition without the release of the dioxygen product, or whether there is another source of electrons. The results are interpreted in terms of the possible existence of an additional water binding component (termed ‘H’) in PS II and a concerted oxidation reaction mechanism for photosynthetic water splitting.